Group members

Research group Virus Immunology
The research group at the UKE and HPI investigates cellular immune responses against viruses that infection humans. "HPI/photographer: Udo Thomas"

Research projects

Research projects focus on the characterization of the molecular mechanisms that enable antiviral NK cells to recognize virus-infected cells and on the impact of sex-specific factors on the development of antiviral immune responses. The working group is involved in a number of DFG and EU-funded joint projects (research networks) .

Antiviral cellular immune responses

The human immune system plays a central role in the control of viral infections. In particular, T cells and NK cells can recognize and kill virus-infected cells. The research group of Prof. Dr. Altfeld examines antiviral T cell and NK cell responses against several viruses, including HIV-1, HCV, and Influenza. In particular, we aim to characterize the pathways by which the immune system recognizes viral infections, and the mechanisms that viruses have developed to evade antiviral immunity. In addition, we have developed in vitro models to analyze the interplay of innate and adaptive immune responses during the course of a viral infection. The aim of these studies is to identify protective immune responses that can be subsequently induced through new vaccination strategies or immunotherapeutic approaches.

Virus detection and immune activation

Human pathogenic viruses can be detected by a number of receptors of the immune system. Cells of the innate immune system express receptors that can identify components of viruses as foreign, and initiate the subsequent antiviral immune response. While activation of innate immunity plays an important role in the initial control of acute viral infections, a persistent activation of the immune system in chronic viral infections (HIV -1, HCV) contributes to viral pathogenesis and pathology (CD4 T cell decline, liver fibrosis). The research group of Prof. Dr. Altfeld investigates the extracellular and intracellular receptors and signaling cascades that lead to the detection of viruses. A particular area of interest is the analysis of Toll-like Receptors (TLRs) and their influence on the pathogenesis of viral infections. Another area of research are the effects of sex hormones on antiviral immunity, and the resulting implications for gender differences in the disease manifestations of viral infections. The aim of these studies is to develop a better understanding of the molecular mechanisms of viral pathogenesis and to develop new immune-modulatory approaches that can reduce persistent immune activation during chronic viral infections.

Molecular Immunology

Short CV

University DegreesBiology, Wesleyan University 1976 (BA)Medicine, University of Tübingen, 1983 (MD)Molceular Biology, University of Hamburg, 1986 (Diploma)

Professor associée at the University of Rouen, France (2006-2007)Visiting scientist at the University of California, San Francisco, CA, USA (1994)Visiting scientist at the DNAX Research Institute of Molecular Biology, Palo Alto, CA, USA (1997)Visiting scientist at The Jackson Lab, Bar Harbor, ME, USA (1991, 1999)

Mentorship Award of the Simon-Claussen Foundation (2009)Research Award of the Werner Otto Foundation (1997)Research Award of the Martini Foundation (1991)

Research projects

The focus of our laboratory is on the molecular characterization of lymphocyte membrane proteins, in particular receptors and enzymes involved in signaling by extracellular nucleotides. We generate monoclonal and single domain antibodies as new research and therapeutic tools. We are interested in ADP-ribosylation as a pathogenic mechanism of bacterial toxins and as a reversible posttranslational modification regulating protein functions. Using genetic immunization and antibody engineering, we strive to develop new tools for combating infections and for treating diseases of the immune system.

Membrane proteins mediate the communication of cells with their environment. They function as receptors for soluble ligands and counter-receptors on other cells, as ion channels, nutrient transporters, and enzymes. The nucleotides NAD and ATP are key metabolites of energy metabolism found in cells from all kingdoms of life. The cell membrane is impermeable to these nucleotides, but they can exit cells via channels or pores gated by mechanical and/or electrical stimuli. During infection and inflammation injured cells release NAD and ATP through the damaged cell membrane. Extracellular NAD and ATP alert cells of the immune system to sites of tissue damage.

Cells of the immune system are equipped with a variety of sensors for these nucleotides (Fig. 1), including ligand-gated ion channels (P2X purine receptors) and nucleotide-metabolizing ecto-enzymes (CD38, CD296). CD296 (ARTs) functions as NAD-sensors and relay information about the levels of extracellular NAD into ADP-ribosylation of cell surface and secreted proteins. The NAD-hydrolyzing ecto-enzyme CD38 restricts the intensity and duration of NAD-signaling in the extracellular space by hydrolyzing NAD. Opening of the P2X7 ion channel is induced by binding of the soluble ligand ATP, or by NAD-dependent ADP-ribosylation. Passage of ions through P2X7 (calcium into the cell, potassium out of the cell) triggers a cascade of downstream events, including protease activation (caspases and ADAMs), externalization of phosphatidylserine (PS), activation of the inflammasome and cell death. Mice that cannot express the mentioned purine receptors or ecto-enzymes show impaired immune responses. The receptors and enzymes of purinergic signaling, thus, present potential targets for new anti-inflammatory or immune-stimulating drugs. In mouse models, activating the ARTC2 > P2X7 axis can enhance anti-tumor responses, while blocking this axis can reduce inflammation in autoimmune diseases.

Antibodies are important tools of research, diagnosis and therapy. Antibodies are raised by immunizing experimental animals with proteins, synthetic peptides, or DNA. In the case of DNA immunizations, the protein encoded by the DNA is produced in its native conformation by the cells of the immunized animal and the induced immune response yields antibodies directed against proteins in native conformation (ADAPINCs) (Fig. 2). Such antibodies are useful in many applications were anti-peptide antibodies fail, e.g. affinity purification, flow cytometry, ELISA, FACS, and functional studies. cDNA immunization is offered as a service via the antibody core facility of the UKE.

Llamas produce unusual antibodies composed only of heavy chains. Their antigen-binding domain (VHH) is readily produced as a soluble recombinant protein, also designated nanobody or single domain antibody (sdAb) (Fig. 3). Nanobodies can form finger like extrusions that block clefts on protein surfaces, such as the active site crevice of enzymes, the ligand binding domain of a receptor, and the receptor binding domain of a virus. Nanobodies have great potential as therapeutics and as imaging agents.

Fig. 3. Schematic diagram of conventional antibodies and of heavy chain only antibodies made by llamas, dromedaries and other camelids.

We have generated enzyme-blocking nanobodies from llamas immunized with different ARTs: the SpvB Salmonella toxin, the binary clostridium difficile toxin CDTa, and the T cell ecto-enzyme ARTC2 (CD296). These nanobodies protect cells from the cytotoxic effects of SpvB, CDT, and ARTC2. In case of ARTC2, the nanobodies effectively block ARTC2 on the cell surface of regulatory T cells and iNKT cells within 10 minutes after intravenous injection. These nanobodies provide an important tool for protecting these regulatory cells from death induced by NAD released during tissue preparation. We have also generated nanobodies against CD38, the major NAD-hydrolyzing ecto-enzyme. CD38 is emerging as a therapeutic target in multiple myeloma and other hematopoertic malignancies. Some of our nanobodies outperform the recently licensed CD38-specific monoclonal antibody Daratumumab (Darzalex) in cytotoxicity vs. hematopoetic cancer cell lines.In cooperation with Ablynx, a Belgian company devoted to developing nanobodies for human therapies, we have generated nanobodies that block or enhance gating of the P2X7 ion channel. The P2X7-blocking nanobodies ameliorated inflammation in mouse models of glomerulonephritis and allergic dermatitis. In endotoxin-treated human blood cells, they effectively blocked LPS/ATP-induced release of the potent pro-inflammatory cytokine IL-1ß. In order to facilitate the generation of new nanobodies, we have cloned the nanobody-encoding IgH locus from llama and successfully transferred an engineered version of this locus to transgenic mice (Fig. 4). Upon immunization, these mice produce nanobody-based heavy chain antibodies that undergo somatic hypermutation and class switch recombination. This novel platform greatly expands our capacity to generate functional nanobodies against interesting targets.

The function of proteins can be regulated via the attachment of chemical moieties. These enzyme-catalyzed modifications, coined posttranslational modifications (PTMs), include phosphorylation, glycosylation, ADP-ribosylation, and the attachment of lipid anchors. ADP-ribosylation is a reversible PTM, in which ADP-ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NAD onto a specific amino acid side chain in a target protein and ADP-ribosylhydrolases (ARHs) remove the ADP-ribose group (Fig. 5). We have determined the 3D structures of a prototype ART and a prototype ARH. The 3D structure of rat ARTC2 resembles a pacman with a wide-open mouth crunching on the substrate NAD. The 3D structure of human ARH3 resembles a pumpkin in which four alpha helices coordinate two magnesium ions at the bottom of the active site crevice.

ADP-ribosylation is used by pathogenic toxins such as Diphtheria, Pertusssis and Clostridial toxins to modulate host protein functions. Toxin-related ARTs are expressed by cells of the immune system. ADP-ribosylation of membrane proteins can be monitored using labeled analogues of NAD. Using 32P-NAD, ADP-ribosylation results in radiolabeling of the target protein. Using etheno-NAD, ADP-ribosylation of target proteins can be detected with a monoclonal antibody directed against etheno-adenosine. This 1G4 antibody can be used to sort cells on the basis of cell surface ART-activity and to purify etheno-ADP-ribosylated proteins.

ARTC2 itself is posttranslationally modified, e.g. by attachment of a GPI lipid anchor (Fig. 1 above). The GPI-anchor targets ARTC2 to lipid rafts, specialized regions of the cell membrane that play an important role in signal transduction, e.g. during activation of T cells by antigen presenting cells. The association of ARTC2 with lipid rafts focuses ARTC2 onto specific target proteins and thereby may regulate the signaling function of raft-associated proteins. ADP-ribosylation of the P2X7 ion channel on arginine residue 125 activates P2X7 to form a non-selective ion-channel, permitting calcium ions to enter the cell and potassium ions to exit the cell. This induces dramatic alterations of the cell membrane, including the externalization of PS, shedding of L-selectin, and formation of membrane blebs.

Group members

Research projects

The impact of pre- and postnatal medical interventions on the developing immune system

The thymus, a primary lymphatic organ, is essential for T cell development and establishment of tolerance in order to avoid autoimmunity later in life. Around birth, thymic function is at its peak and therefore, events affecting the thymus during this sensitive period may have serious consequences for the child’s immunity.

Recent findings suggest that impairment of the thymus function during this highly vulnerable time around birth might increase the risk for autoimmune and chronic diseases like type I diabetes, allergies or asthma, later in life. Interestingly, the prevalence of autoimmune and chronic diseases increased dramatically during the past decades in industrialized countries. This trend can’t be explained exclusively by genetic factors, indicating that early-life environmental factors play a crucial role in shaping immune maturation.

Our research activities focus on the thymus and the development of the immune system, particularly the impact of early-life medical interventions (e.g. antenatal corticosteroid treatment and thymectomy) on the development of autoimmunity and other chronic diseases.

Research project: The impact of pre- and postnatal medical interventions on the developing immune system

Characterization of the adenine-metabolising ectoenzymes and purinergic rezeptors

The extracellular adenine nucleotides and –nucleosides (AN) ATP and NAD and their metabolites play key roles in the modulation of the immune response. We are interested in the regulation of the ectoenzymes involved in the generation and destruction of AN, their role in different pathways of the immune response, and in the outcome upon activation of purinergic receptors by AN.

The cell membrane ectonucleotidase CD39 hydrolyzes ATP and ADP to AMP, acting in concert with CD73 to generate adenosine. By this process, a pro- inflammatory molecule, ATP, is degraded to generate the anti-inflammatory adenosine. In mice, regulatory T cells (Tregs) express both ectoenzymes, and use this pathway to suppress the immune response. In humans, however, only some Tregs express CD39, and most of them do not display CD73 on the cell surface. Tregs expressing CD39 are powerful suppressors of T cell proliferation and, especially, of the production of inflammatory cytokines. We have shown that expression of CD39 on Tregs is primarily genetically determined, and this may determine interindividual differences in the control of inflammatory responses. In addition, T cell activation results in further upregulation of CD39. Much less is known on the modulation of CD73 and its consequences in human disease, although adenosine tightly controls immune cell performance by binding to P1 receptors on different cell types.

High pericellular concentrations of ATP trigger P2X receptor activation. Binding of ATP to the nucleotide-gated ion channel P2X7 results in influx of Ca2+, leading to inflammasome activation and release of IL-1β and IL-18, activation of metalloproteases and cell death, altogether contributing to an inflammatory environment. Thus, modulation of the ATP-adenosine axis constitutes an attractive intervention target. Since immune regulation must be strenghtened or reduced according to the type of disease, understanding the regulation of the ectoenzymes CD39 and CD73, and of P1 and P2 receptors is a crucial first step for the development of novel therapies. We plan to use chemical and biological inhibitors as well as knock-out systems for assessing the cell-specific role of these enzymes and receptors in the modulation of immunity and to evaluate the possibility for intervention.

elucidating how and when ATP is released from immune cells, and how it is metabolized to adenosine on the outside.

Because of our close connection to the Clinical Immunodiagnostics Laboratory our group is also interested in evaluating and improving immunodiagnostic methods suitable for routine use. Here our focus is currently on the development of flow cytometry protocols for the characterization of immune cells from patients. We also provide assistance and immunodiagnostic services for research projects that need to measure immunological parameters.

Extracellular nucleotides as regulators of lymphocyte function

Besides their metabolic importance inside cells, adenosine triphosphate (ATP) and other adenine nucleotides fulfill important roles outside of cells as autocrine and paracrine signal mediators. ATP is abundant within cells (3-10 mM), but under steady state conditions it is barely detectable in the extracellular milieu (low nanomolar conentrations). However, cells can actively secrete ATP for signaling purposes, or it can be passively released from cells upon cell damage. Once released into the extracellular space, ATP acts on ionotropic P2X or the metabotropic P2Y receptors present on the cell surface. While P2Y receptors belong to the family of G protein-coupled receptors (GPCRs), P2X receptors are ATP-gated cation channels, which among other actions can mediate the influx of calcium ions (Ca2+) into the cell. Outside the cell, ATP is degraded by the combined actions of ecto-nucleoside triphosphate diphosphohydrolases (ENTPDs) such as CD39 and 5’-nucleotidases like CD73, generating adenosine (ADO), which is a ligand for another family of GPCRs, the P1 receptors.

Regulating the balance between extracellular ATP and ADO concentrations is an important mechanism for the regulation of inflammation. The release of ATP is an ancient, evolutionarily conserved danger signal heralding tissue damage, and thus alerting immune cells to initiate or augment an inflammatory reaction. This pro-inflammatory signal is counteracted by the degradation of ATP to ADO. Especially, stimulation of the A2A and A2B P1 receptors acts on many immune cells as a strong inhibitory (and thus anti-inflammatory) signal.

In a similar fashion, extracellular nicotinamide adenine dinucleotide (NAD) also initiates signaling cascades by serving as a substrate either for the ADP-ribosyltransferase ARTC2 or for the ecto-NADase CD38. On mouse T lymphocytes, which carry ARTC2, a prominent target for ADP-ribosylation is the P2X7 ion channel, which is activated by this modification. CD38 uses NAD and NADP to generate calcium-mobilizing second messengers such as cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) (Fig. 1).

Infection Immunology

The section „Infection Immunology“ headed by Prof. Dr. Hans-Willi Mittrücker is focused on the regulation of immune responses against pathogens and on the function of T cells in autoimmune kidney diseases.

Group members

Research projects

CD4 and CD8 T cells play a central role in immune responses against pathogens, yet these cells are also crucial for the formation of autoimmune diseases. We use different mouse models for bacterial infection and for autoimmune kidney diseases to characterise the function and regulation of T cells. Main focuses of research are:

The function and regulation of CD4 and CD8 T cells during infection with intracellular bacteria. For these studies, we use infection models for Listeria monocytogenes and Salmonella typhimurium.

The role of the transcription factor Interferon Regulatory Factor 4 (IRF4) in activation and differentiation of T cells.

The regulation of immune responses by Interleukin-6, in particular, the impact of different Interleukun-6 signalling pathways on activation and differentiation of immune cells.

Autoimmune kidney diseases and the role of T cells in formation and regulation of immune response against components of the glomerulus. Studies are conducted in mouse models for anti-glomerular basement nephritis.

The role of adenine nucleotides, ectoenzymes and purinegic receptors for the regulation of T cells.

Selected publications

Function and regulation of T cells during infection with intracellular bacteria